Introduction to Histology PDF

INTRODUCTION TO HISTOLOGY Histology Microtome Histology - Greek “histos” meaning “.ssue” and “logos” meaning “study of” - Study of the.ssues of the body and how these.ssues are arranged to cons.tute organs. -...

INTRODUCTION TO HISTOLOGY Histology Microtome Histology - Greek “histos” meaning “.ssue” and “logos” meaning “study of” - Study of the.ssues of the body and how these.ssues are arranged to cons.tute organs. - Involves all aspects of.ssue biology, with the focus on how cells' structure and arrangement op.mize func.ons specific to each organ. - Two interac.ng components: cells and extracellular matrix (ЕСМ). Prepara;on of Tissues for Study - The.ssue block is then mounted onto the microtome for trimming and cu`ng: o Trimming – trims large por.ons o CuGng – real cu`ng cuts to from “.ssue ribbons” § Tissue ribbons are then placed 2 ways to acquire ;ssues: in a water bath and fished out 1. Biopsy – from alive specimen with a microscope slide 2. Autopsy – from dead specimen Step Descrip;on Fixa.on Uses a fixa.ve to preserve the.ssue in its life-like state, which is usually formalin. Dehydra.on Removes water from the.ssue by exposing it to increasing alcohol concentra.on. This is because water facilitates decay in a.ssue. Staining Usually 30%, 50%, 70%, and 90% - Opposite abracts ethanol Acidic Structures – Basic dye Basophilic Clearing The.ssue is cleared of the alcohol (e.g. nucleus, DNA) used in the previous step through Basic Structures – Acidic dye Acidophilic the use of xylene. (e.g. cytoplasm, major Infiltra.on A\er clearing, the.ssues have holes basic protein in in them. They must be exposed to an granules of infiltra.ng agent in order to support eosinophils) the.ssue. - Common dyes: Hematoxylin (basic) & eosin (acidic) Infiltra.ng agent is usually paraffin wax. This step is done in high Microscopy temperature (52°C–60°C) which is 1. Light Microscopy the mel.ng point of paraffin. Bright-Field Microscopy Embedding The.ssue needs to be supported in Fluorescence Microscopy a medium to help in cu`ng, which is Phase-Contrast Microscopy usually paraffin wax. Dark-Field Microscopy Confocal Microscopy Once embedded, it will then be Polarizing Microscopy called a “.ssue block”. 2. Electron Microscopy Transmission Electron Microscopy Scanning Electron Microscopy  - Expensive and does not age well (fluorescence Light Microscopy fades) 1. Bright-Field Microscopy - Stained prepara.ons are examined by means of ordinary light that passes through the specimen. - Resolving power o Defined as the smallest distance between two par.cles at which they can be seen as separate objects. o Ability of a microscope lens/op.cal system to produce separate images of closely posi.oned objects o 0.2 µm (ribosomes, membrane, ac.n filaments are smaller than 0.2 µm and 3. Phase-Contrast Microscopy cannot be seen in this microscope) - Based on the principle that light changes its speed when passing through cellular and extracellular structures with different refrac.ve indices. - Uses a lens system that produces visible images from transparent objects - Enables examina.on of unstained cells and.ssues and is especially useful for living cells. - A bright-field microscope can be modified into a Phase-Contrast with the use of filter and op.cs - Can observe actual cell processes (phagocytosis) - Types: a. Interference Microscope o Allows quan.fica.on of.ssue mass - Condenser collects and focuses light and b. Differen;al Interference Microscope eliminate the object o Using Nomarski op.cs - A dual-lens system, where the image would o Useful for assessing surface proper.es first be enlarged in the objec.ves then of cells and other biological objects projected to the ocular which will then be BF Bright Field PC Phase-contrast DI Differential Interference Microscope magnified by the eyepiece and projected into the re.nas. - Objec;ve is the first to magnify 2. Fluorescence Microscope - Tissue sec.ons are usually irradiated with ultraviolet (UV) light and the emission is in the visible por.on of the spectrum. - The fluorescent substances appear brilliant on a dark background. - Fluorescence o Phenomenon wherein certain cellular 4. Dark-Field Microscopy substances are irradiated by light of a - No direct light from the light source is gathered proper wavelength, they emit light by the objec.ve lens with a longer wavelength. - Only light that has been scabered or diffracted o UV light excites the fluorescent dye by structures in the specimen reaches the and makes it jump to a higher energy objec.ve level. Higher energy levels are unstable - Equipped with a special condenser that therefore it must go back to its original illuminates the specimen with strong, oblique level, and as it goes back it emits the light excess energy in the form of fluorescent light.  - Field of view appears as dark background on which small par.cles in the specimen reflect some light into the objec.ve appear bright - Spirochetes in syphilis Light exits in polarizing filter vibra.ng in only one direc.on Second filter (perpendicular) does not allow light to pass through If.ssue has birefringence capability (created by repe..ve structures in its axis) they would vibrate the light and create a bright structure 5. Confocal Microscopy against the background - Combines components of a light op.cal - E.g. Collagen Fibers microscope with a scanning system to dissect à specimen op.cally. - Uses (1) a small point of high-intensity light, o\en from a laser, and (2) a plate with a pinhole aperture in front of the image detector. - Point light source, the focal point of the lens, and the detector's pinpoint aperture are all op.cally conjugated or aligned to each other in the focal plane (confocal). and unfocused light does not pass through the pinhole. - Reduces stray light to increase contrast (affects resolving power) Electron Microscopy - Based on the interac.on of.ssue components with beams of electrons. - The wavelength in the electron beam is much shorter than that of light, allowing a 1000-fold increase in resolu.on. - 2 Kinds: o TEM o SEM 1. Transmission Electron Microscopy - An imaging system that permits resolu.on 6. Polarizing Microscopy around 3 nm. - Uses the fact that highly ordered molecules or - Magnifica.ons of up to 400,000.mes to be arrays of molecules can rotate the angle of the viewed in detail plane of polarized - Uses the interac.on of a beam of electrons - Birefringence with a specimen to produce an image. o ability to rotate the direc.on of - Cryofracture and freeze etching vibra.on of polarized light o a special method of sample o usually in ac.n filaments and crystals prepara.on for transmission electron microscopy o It is especially important in the study of membranes.  - Immunofluorescence - 2 Types: Direct Immunofluorescence – uses an.bodies with fluorescent tags that will fluoresce when they interact with the an.gen Indirect Immunofluorescence – uses primary and secondary an.body o Primary an.body interacts Cathode (tungsten filament) emits an electron with the an.gen to go to the anode, which will then pass o Secondary an.body, which through the condenser lens which focuses all carries the tag, comes a\er the electromagne.c beams to the circular coils the primary interacts with the Some electrons pass through, and some do not an.gen and binds to the Images are flat but detailed primary an.body itself. Electron-loosened area (bright) – electrons (Basically, an an.body against passed through the primary an.body) Electron-dense area (dark) – absorbed or deflected electrons 2. Scanning Electron Microscopy - The electron beam does not pass through the specimen but is scanned across its surface. - The surface of the specimen is first dried and spray-coated with a very thin layer of heavy metal (o\en gold) through which electrons do 3. Hybridiza;on Techniques not pass readily. - A method of localizing messenger RNA (mRNA) - When the beam is scanned from point to point or DNA by hybridizing the sequence of interest across the specimen, it interacts with the metal to a complementary strand of a nucleo.de atoms and produces reflected electrons or probe secondary electrons emibed from the metal - In situ hybridiza;on - Electron interacts with the surface o Binding of the nucleo.de probe to the - Results are 3D but less detailed DNA or RNA sequence of interest is performed within cells or.ssues, such as cultured cells or whole embryos. o E.g.: Viruses do not have double-helix DNA, therefore complementary strands (which have fluorescent tag that light up once the strands bind) are created which will bind if the virus is present Other Methods 1. Enzyme Histochemistry - A method for localizing cellular structures using a specific enzyma.c ac.vity present in those structures. - Green probe (Chromosome 13) - Orange probe (Chromosome 21) 2. Immunochemistry - A highly specific interac.on between molecules is that between an an.gen and its an.body Interpreta;on of Structures in Tissue Sec;ons